Predominant expression and cellular distribution of fish Agr2 in renal collecting system.

Anterior gradient 2 (Agr2) genes encode secretory proteins, and play significant roles in anterior-posterior patterning and tumor metastasis. Agr2 transcripts were shown to display quite diverse tissue distribution in different species, and little was known about the cellular localization of Agr2 proteins. In this study, we identified an Agr2 homologue from gibel carp (Carassius auratus gibelio), and revealed the expression patterns and cellular localization during embryogenesis and in adult tissues. The full-length cDNA of CagAgr2 is 803 nucleotides (nt) with an open reading frame of 510 nt encoding 169 amino acids. The Agr2 C-terminus matches to the class I PDZ-interacting motif, suggesting that it might be a PDZ-binding protein. During embryogenesis, CagAgr2 was found to be transcribed in the mucus-secreting hatching gland from tailbud stage and later in the pharynx region, swim bladder and pronephric duct as revealed by RT-PCR and whole mount in situ hybridization. In the adult fish, its transcription was predominantly confined to the kidney, and lower transcription levels were also found in the intestine, ovary and gills. To further localize the Agr2 protein, the anti-CagAgr2 polyclonal antibody was produced and used for immunofluorescence observation. In agreement with mRNA expression data, the Agr2 protein was localized in the pronephric duct of 3dph larvae. In adult fish, Agr2 protein expression is confined to the renal collecting system with asymmetric distribution along the apical-basolateral axis. The data provided suggestive evidence that fish Agr2 might be involved in differentiation and secretory functions of kidney epithelium.

[Screen of differentially expressed genes between gastrula embryos and tail bud embryos in gynogenetic gibel carp (Carassius auratus gibelio)].

Suppression subtractive hybridization (SSH) cDNA plasmid libraries were constructed between gastrula embryos and tail bud embryos in gynogenetic gibel carp (Carassius auratus gibelio). 739 and 816 PCR positive clones were respectively selected to perform dot blot, and 72 dot blot positive clones and 98 dot blot positive clones were obtained from the SSH plasmid libraries specific for gastrula embryos and tail bud embryos. Sequencing analysis and database searches indicated that there were 19 known genes and 31 unknown/cDNA fragments in the sequenced 72 dot blot positive clones specific for gastrula embryos, and 52 known genes and 37 unknown cDNA fragments in the sequenced 98 dot blot positive clones specific for tail bud embryos. Moreover,specific expressions of partial genes were further confirmed by virtual Northern blots and RT-PCR. The screen of these differentially expressed genes will help us to understand the molecular mechanism in gibel carp embryogenesis.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp.

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.